Genotype‐phenotype correlations in multiple lesions of familial cerebral cavernous malformations concerning phosphatidylinositol 3‐kinase catalytic subunit alpha mutations

Our study suggested that in familial multiple cerebral cavernous malformations (CCMs), symptomatic intracerebral haemorrhage (ICH) lesions and dot-sized lesions harbour distinct clinical features and genotypes. Beva-cizumab, a vascular endothelial growth factor (VEGF) inhibitor, might be a potential therapeutic agent for familial CCMs.

Vitro and vivo experiments both indicated that PI3K-VEGF pathway activation plays a vital role in the genotype- phenotype correlations.We hypothesized that VEGF inhibitors might restrain bleeding.Bevacizumab, a VEGF inhibitor, is an effective therapy for cancers.To investigate the effect of bevacizumab, P60 Krit1 iECKO mice were injected with AAV-PIK3CA H1047R via retro-orbital sinus and bevacizumab through the tail vein, and the control mice were injected with AAV-PIK3CA H1047R and vehicle alone.Then, the mice were subjected to MRI and histological examination at P90 (Figure 4A).Surprisingly, bevacizumab treatment inhibited ICH formation (Figure 4B,C).Furthermore, haematoxylin and eosin staining revealed that bevacizumab dramatically decreased the number of CCM lesions and prevented the formation of larger CCMs (Figure 4D-F).
Our study has several limitations.First, for ethical considerations, we cannot obtain more surgical specimens of dot-sized lesions in the familial CCMs; second, some paired blood samples were unavailable in FFPE samples and the mutation type of surgical samples (germline or somatic) was identified by minor allele frequency (MAF); finally, for the suboptimal quality of sample DNA, the MAF of CCM germline mutation were below 50%, particularly in the FFPE samples.
Here, we identified that in familiar CCMs, the two subgroup lesions have distinct genotype-phenotype correlations concerning PIK3CA mutations, which implies differentiated management strategy might be taken.Bevacizumab, as a promising therapeutic agent for familial CCMs, requires further prospective clinical study to evaluate its safety and efficacy due to the risk of complications such as hypertension and cardiac ischemia. 10

A U T H O R C O N T R I B U T I O N S
Jiguang Wang and Yong Cao conceived and designed the study; Jie Wang contributed to clinical data collection, samples collection, vivo and vitro experiment, data analysis and wrote the manuscript; Jihong Tang and Yingxi Yang contributed to bioinformatics analysis; Ran Huo and Hongyuan Xu contributed to experimental design and implementation; Yingfan Sun, Yuming Jiao, Qiheng He, Shaozhi Zhao and Qifeng Yu helped with collecting the samples.Shuo Wang, Jizong Zhao and Yong Cao performed the operation and provided CCMs samples and control specimens; Jiguang Wang supervised bioinformatics studies.Jiguang Wang and Yong Cao provided overall oversight of the research.

A C K N O W L E D G E M E N T S
We thank Professor Xiangjian Zheng (Department of Pharmacology, Tianjin Medical University, China) and Xi Yang (Department of Pharmacology, Tianjin Medical University, China) for kindly providing transgenic mice and experimental assistance in our study.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

E T H I C S S TAT E M E N T
The study was approved by the Institutional Review Board and the Ethics Committee of Beijing Tiantan Hospital.(KY2017-035-02).The Animal Welfare and Ethics Committee of Beijing Neurosurgical Institute Laboratory approved all animal ethics and protocols.

F I G U R E 1
Symptomatic intracerebral haemorrhage (ICH) lesions and dot-sized lesions in familial cerebral cavernous malformations (CCMs) have distinct natural courses.(A) The classification of multiple lesions in familial CCMs.White arrows (ICH lesions) and black arrows (dot-sized lesions).(B) Total lesions, follow-up months, haemorrhage event and surgical resection of enrolled patients.A patient with two ICH lesions was resected a lesion at first hospitalization.(C) The Kaplan-Meier curve showing the cumulative rate of haemorrhage events between ICH lesions and dot-sized lesions (black arrows) (Log-rank test p < .001).(D) Representative follow-up magnetic resonance imaging (MRI) of ICH lesions (white arrows) and dot-sized lesions (black arrows).(E) Representative follow-up MRI of dot-sized lesions (black arrows) after surgical resection of ICH lesion.Postop, postoperative.

F I G U R E 2
Mutational landscape of familial cerebral cavernous malformations (CCMs).(A) Summary of germline and somatic mutations in 18 two subgroup lesions of CCM (three dot-sized lesions and 15 intracerebral haemorrhage [ICH] lesions) by whole exome sequencing (WES) and/or droplet digital polymerase chain reaction (ddPCR).(B) ddPCR showed ranked mutation allele frequencies of phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutation in the study.Mutational incidence of PIK3CA mutation detected by ddPCR was displayed by a pie chart.(C) Enrichment of genotype in ICH and dot-sized lesions.*, p < .05.
This study is funded by Genomics Platform Construction for Chinese Major Brain Disease-AVM (PXM2019_026280_000002-AVM); Beijing Advanced Innovation Center for Big Data-based Precision Medicine (PXM2020_014226_000066). Research in Wang lab is supported by RGC grants (16102522, C6021-19EF), ITC grant (ITCPD/17-9), a project of Hetao Shenzhen-Hong Kong Science and Technology Innovation Cooperation Zone (HZQB-KCZYB-2020083), and the Padma Harilela Professorship.D ATA AVA I L A B I L I T Y S TAT E M E N T Deidentified data that are not published within this article will be made available to any qualified investigator upon request.
1,2,6Correspondence Jiguang Wang, Division of Life Science, Department of Chemical and Biological Engineering, the Hong Kong